Volume 3, Issue 6, Pages 850-856 (June 2001) In Vivo Treatment of Hemophilia A and Mucopolysaccharidosis Type VII Using Nonprimate Lentiviral Vectors  Colleen S. Stein, Yubin Kang, Sybille L. Sauter, Kay Townsend, Patrick Staber, Todd A. Derksen, Inês Martins, Jiahua Qian, Beverly L. Davidson, Paul B. McCray  Molecular Therapy  Volume 3, Issue 6, Pages 850-856 (June 2001) DOI: 10.1006/mthe.2001.0325 Copyright © 2001 American Society for Gene Therapy Terms and Conditions

FIG. 1 Histochemical detection of β-glucuronidase after FIVβgluc injection. (a) Negative control liver from an uninjected MPS VII mouse. (b) Liver of a mouse 3 weeks after injection of 6 × 107 TU of FIVβgluc (Experiment 1, mouse 2, Table 2). (c) Liver of a mouse 11 weeks after injection of 6 × 107 TU of FIVβgluc (Experiment 1, mouse 3, Table 2). (d) Liver from a mouse injected with 3 × 108 TU of FIVβgluc 3 weeks earlier (Experiment 2, mouse 1, Table 2). (e–h) Spleen, lung, kidney, and heart, respectively, from a mouse injected with 3 × 108 TU of FIVβgluc 3 weeks previously (Experiment 2, mouse 1, Table 2). Original magnification 20×. Molecular Therapy 2001 3, 850-856DOI: (10.1006/mthe.2001.0325) Copyright © 2001 American Society for Gene Therapy Terms and Conditions

FIG. 2 Histopathology of untreated or FIVβgluc-injected MPS VII mice. (a) Liver of a 20-week-old untreated MPS VII mouse shows abundant lysosomal storage (clear vacuoles). (b) Liver of an MPS VII mouse, 3 weeks after injection of 3 × 108 TU of FIVβgluc (age at injection was 22 weeks) (Experiment 2, mouse 1, Table 2). (c) Liver of an MPS VII mouse, 11 weeks after injection of 6 × 107 TU of FIVβgluc (age at injection was 7 weeks) (Experiment 1, mouse 3, Table 2). (d) Spleen of a 24-week-old untreated MPS VII mouse. (e) Spleen of an MPS VII mouse, 3 weeks after injection of 3 × 108 TU of FIVβgluc (age at injection was 22 weeks) (Experiment 2, mouse 1, Table 2). Original magnification 100×. Molecular Therapy 2001 3, 850-856DOI: (10.1006/mthe.2001.0325) Copyright © 2001 American Society for Gene Therapy Terms and Conditions

FIG. 3 Tissue distribution of vector provirus and transcripts after injection of FiVβgluc. DNA and RNA were isolated from tissues of MPS VII mice 3 weeks after intravenous injection of 3 × 108 IU of FiVβgluc, or from tissues of an untreated MPS VII mouse, and analyzed by PCR or RT-PCR. Lanes from individual FiVβgluc-injected mice are labeled 1, 2, and 3, and lanes from an uninjected MPS VII control mouse are labeled c. The left lane of each gel shows size markers, with band sizes labeled in base pairs. (a) The top gel shows PCR analysis for a 538-bp human β-glucuronidase cDNA sequence. The right lanes present PCR results using the β-glucuronidase vector plasmid as template, serially diluted into DNA from a naive MPS Vii mouse (numbers above the lanes indicate the ratio of plasmid copies per diploid genome for each sample). The bottom gel shows PCR analysis for a 575-bp murine β-glucuronidase genomic sequence. This PCR serves as a positive control for the integrity of the DNA samples. (b) RT-PCR analysis for transcripts containing a 538-nucleotide human β-glucuronidase sequence (top gel) or a 335-nucleotide β-actin sequence (bottom gel). The “No RT” control lane (top gel) shows results from an RT-PCR performed in the absence of reverse transcriptase, using RNA isolated from liver sample 1. Molecular Therapy 2001 3, 850-856DOI: (10.1006/mthe.2001.0325) Copyright © 2001 American Society for Gene Therapy Terms and Conditions